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Imaging of Monoaminergic and Cholinergic Vesicular Transporters in the Brain
Kirk A. Frey,*t$ Donald M. Wieland," and Michael R. Kilbourn" Departments of "Internal Medicine (Nuclear Medicine) and tNeurology and *The Mental Health Research Institute The University of Michigan Ann Arbor, Michigan 48 I09
Imaging of Monoaminergic and Cholinergic Vesicular Transporters in the Brain Studies of chemically defined neurons may offer important neurochemical perspectives on normal aging and on age-associated neurodegenerative diseases. Conditions including Parkinson's disease (PD) and Alzheimer's disease (AD), for example, share age-associated increasing incidences and may be defined by pathologic losses of neurons and synapses in specific brain regions, or involving select chemical neuronal classes. The pathophysiologies of most neurodegenerative disorders are unknown, despite detailed descriptions of the ultimate neuropathologic effects of the disease processes. Postmortem analyses, however, may be confounded by the intrusions of secondary, disease-compensatory or treatment-induced changes. Thus, in vivo investigations conducted early in the course of neurodegenerative diseases offer unique opportunities to determine the initial and most specific neurochemical alterations. Furthermore, in vivo neurochemical measures may have the potential to distinguish diseasemodifying from symptomatic therapies. In this instance, objective, quantitative markers of neuronal or synaptic integrity, targeting the involved cell types and brain regions, may offer considerable advantage over clinical examinations or other indirect measures in tracking the course and progression of neuropathology. With these goals and applications in mind, our laboratories have focused on the development of in vivo imaging markers of the dopaminergic and cholinergic neurons and synapses of the human brain, employing positron emission tomography (PET) or single photon emission tomography (SPECT) of radiolabeled neurochemical tracers. Among the possible biochemical markers of specific neurons and synapses, including neurotransmitter synthetic enzymes, plasma membrane uptake transporters, and neurotransmitter receptors, a quantitative relationship between neuron or synapse integrity and the level of marker expression in macroscopic tissue samples is difficult to establish. The activities and expressed protein levels of these markers are well recognized to undergo compensatory regulation in response to therapeutic drugs and in response to disease conditions. Recent molecular and pharmacologic advances now permit the study of an additional class of neuronal markers, the distinct vesicular neurotransmitter transporters Advancer in Pharmacology, Volume 42 Copyright (C 1998 by Academic Press. All rights ot reproduction in any form reserved 1054-3589/98 $25.00
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in monoaminergic and cholinergic neurons. Although neurotransmitter release from synaptic vesicles is a regulated process, it has been suggested that a major aspect of this regulation may reside in the partitioning of vesicles between active (cycling) and reserve (bound) pools in nerve terminals (1).We hypothesized that vesicular markers not distinguishing these states might serve as quantitative indicators of synaptic density, with minimal or no influence of regulatory changes. Measures of the vesicular neurotransmitter transporters may, thus, provide unique insights into conditions that alter synaptic regulation versus neuronal integrity. We synthesized both unlabeled and radioligand series targeting the neuronal vesicular monoamine transporter type-2 (VMAT2)or the vesicular acetylcholine transporter (VAChT). Ligand-binding specificities were established in vitro in direct binding and competition assays and after selective lesions of dopaminergic or cholinergic projection pathways. The possible regulation of VMAT2 and of VAChT by drugs that alter synaptic transmission were also studied. Single photon- or positron-emitting ligand analogues were then developed for in vivo SPECT and PET imaging of VMAT2 and VAChT. In vitro studies identified specificVMAT2 binding either with [3H]methoxytetrabenazine (2) or with stereochemically resolved ( +)-[3H]dihydrotetrabenazine (DTBZ). Binding of both ligands was saturable, reversible, and of high affinity ( K ~ 4s nM and 1.5 nM, respectively) to an apparently homogeneous population of binding sites on intact, slide-mounted brain sections. The brain regional pattern of binding was consistent with the known distributions of dopaminergic, noradrenergic, and serotonergic neurons and their projections. After 6-hydroxydopamine lesions, linear correlation of striatal VMAT2 binding and nigral tyrosine hydroxylase-immunoreactive neuron density was observed over a wide range of lesion severity (2). Furthermore, VMAT2 binding was not affected by 2-wk pretreatment of animals with a variety of drugs leading to regulatory changes in dopamine receptors or in the synaptic membrane dopamine transporter ( 3 ) .In vivo studies in rodents revealed cerebral biodistributions of the tracers paralleling the in vitro pattern of specific VMAT2 binding and additionally confirmed lack of interfering effects of nonvesicular ligand, dopaminergic drugs. Chromatographic analysis after the injection of DTBZ revealed only unchanged tracer in brain, permitting use of total tissue tracer levels in subsequent tracer kinetic analyses. Human imaging experiments employing PET and ["CIDTBZ revealed separable effects of tracer delivery (blood-to-brain transport) and VMAT2 binding on the cerebral time courses of activity after intravenous injection (4). We detected significant age-associated losses of striatal VMAT2 binding in normal subjects, corresponding to linear 0.8% per year losses of VMAT2 from the putamen. Patients with PD demonstrated substantial reductions in VMAT2 in the putamen and more minor losses in the caudate nucleus in the early stages. Patients with severe, advanced PD demonstrated near-complete losses of VMAT2 binding throughout the striatum. Patients with multiple-systems atrophy and some patients with sporadic olivopontocerebellar atrophy also demonstrate reduced striatal VMAT2 binding, supporting prior hypotheses of phenotypic overlap between these two conditions.
Imaging of Vesicular Transporters in the Brain
27 I
Initial in vitro analyses of the VAChT, based on [3H]vesamicol binding, revealed multiple sites of interaction for the ligand. Specific VAChT binding was identified for benzovesamicol-derivative ligands, including methylaminobenzovesamicol (MABV) and 5-iodobenzovesamicol (IBVM). Similarly to the VMAT2, VAChT expression was not regulated by drug treatments affecting ACh turnover and release. After fornix lesions, virtually complete loss of hippocampal VAChT binding was observed in in vitro VAChT binding assays. Intravenous injections of ['*'I]IBVM led to initial uptake in proportion to cerebral blood flow, but delayed retention in a pattern consistent with the in vitro distribution of VAChT, including heavy labeling of cholinergic nuclei and projections ( 5 ) .Fimbrial lesion reduced in vivo retention of ['2SI]IBVMto background levels, confirming the specificity of IBVM biodistribution. Human brain imaging of VAChT in vivo was developed on the basis of SPECT employing ['231]IBVM.After intravenous injections, tracer entered the brain readily and continued to rise over 12-18 hr in the striatum. The brain regional tracer distribution at 24 hr postinjection paralleled the VAChT distribution observed in in vitro assays. Tracer kinetic analyses confirmed close correlation of in vivo ['231]IBVMbinding to VAChT and the levels of delayed tracer retention, permitting simplified experimental determinations of immediate tracer uptake and 24-hr delayed retention in subsequent clinical studies of aging and dementing disorders. In contrast to striatal VMAT2, cerebral VAChT binding was not significantly affected by aging. Unexpectedly, however, VAChT binding showed relatively minor losses in the cerebral cortex of patients with AD ( 6 ) ,despite extensive prior reports of reduced cortical presynaptic cholinergic markers such as choline acetyltransferase (CAT)and acetylcholinesterase activities. There are apparent distinctions between AD patients with presenile versus senile onset of symptoms, the former demonstrating apparently greater VAChT reductions. In addition, PD patients with dementia reveal reduced cortical VAChT, in keeping with postmortem findings of concomitant AD changes in approximately half of demented PD patients. We have confirmed the SPECT VAChT findings in postmortem in vitro assays of AD (7).Samples of temporal neocortex reveal nonsignificant, approximately 15% losses of VAChT binding, yet have over 40% losses of CAT activity in parallel assays. Both VAChT and CAT are significantly reduced in the hippocampal formation of AD, but again, the VAT reduction is less than half of the magnitude of CAT change. These findings suggest the possibility of persistent cholinergic nerve terminals in AD cortex, but with reduced or absent CAT expression. Surviving cholinergic terminals may, thus, provide an attractive target substrate for the possible treatment of the cholinergic lesion in AD with neurotrophic factors that might restore CAT and other obligatory transmitter synthetic activities. In summary, vesicular neurotransmitter transporters appear to provide measures of presynaptic cholinergic and monoaminergic neuronal integrity, unaffected by use- or drug-induced regulatory changes. The roles of cholinergic and monoaminergic neurons in neurodegenerative diseases and their responses to potential disease-modifying therapies can now be specifically studied in vivo with PET and SPECT measures.
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Acknowledgments The studies reported in this work were supported in part by U.S. Public Health Service grants AGO8671 and MH47611 from the National Institute of Aging and the National Institute of Mental Health, respectively.
References 1 . Greengard, P., Valtorta, F., Czernik, A. J., and Benfenati, F. (1993).Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 780. 2. Vander Borght, T. M., Sima, A. A. F., Kilbourn, M. R., Desmond, T. J., Kuhl, D. E., and Frey, K. A. (1995). [’HIMethoxytetrabenazine: A high specific activity ligand for estimating monoaminergic neuronal integrity. Neuroscience 68, 995-962. 3. Vander Borght, T. M., Kilbourn, M. R., Desmond, T. J., Kuhl, D. E., and Frey, K. A. The vesicular monoamine transporter is not regulated by dopaminergic drug treatments. Eur. J. Pharmacol. 294, 577-583. 4. Frey, K. A., Koeppe, R. A., Kilbourn, M. R., Vander Borght, T. M., Albin, R. L., Gilman, S., and Kuhl, D. E. (1996). Presynaptic monoaminergic vesicles in Parkinson’s disease and normal aging. Ann. N e w o l . 40, 873-884. 5 . Jung, Y-W., Frey, K. A., Mulholland, G. K., del Rosario, R., Sherman, P. S., Raffel, D. M., Van Dort, M. E., Kuhl, D. E., and Wieland, D. M. (1996). Vesamicol receptor mapping of brain cholinergic neurons with radioiodine labeled positional isomers of benzovesamicol. J. Med. G e m . 39, 3331-3342. 6. Kuhl, D. E., Minoshima, S., Fessler, J. A., Frey, K. A., Foster, N. L., Ficaro, E. P., Wieland, D. M., and Koeppe, R. A. (1996).In vivo mapping of cholinergic terminals in normal aging, Alzheimer’s disease and Parkinson’s disease. Ann. Neurol. 40, 399-410. 7. Murrnan, D. L., Desrnond, T. J., Higgins, D. S., Hermanowicz, N. S., Penney, J. B. Jr., and Frey, K. A. (1994).Autoradiographic quantification of the vesamicol receptor in Alzheimer’s disease. Neurology 44, (Suppl. 2), A389.
Imaging of Monoaminergic and Cholinergic Vesicular Transporters in the Brain Studies of chemically defined neurons may offer important neurochemical perspectives on normal aging and on age-associated neurodegenerative diseases. Conditions including Parkinson's disease (PD) and Alzheimer's disease (AD), for example, share age-associated increasing incidences and may be defined by pathologic losses of neurons and synapses in specific brain regions, or involving select chemical neuronal classes. The pathophysiologies of most neurodegenerative disorders are unknown, despite detailed descriptions of the ultimate neuropathologic effects of the disease processes. Postmortem analyses, however, may be confounded by the intrusions of secondary, disease-compensatory or treatment-induced changes. Thus, in vivo investigations conducted early in the course of neurodegenerative diseases offer unique opportunities to determine the initial and most specific neurochemical alterations. Furthermore, in vivo neurochemical measures may have the potential to distinguish diseasemodifying from symptomatic therapies. In this instance, objective, quantitative markers of neuronal or synaptic integrity, targeting the involved cell types and brain regions, may offer considerable advantage over clinical examinations or other indirect measures in tracking the course and progression of neuropathology. With these goals and applications in mind, our laboratories have focused on the development of in vivo imaging markers of the dopaminergic and cholinergic neurons and synapses of the human brain, employing positron emission tomography (PET) or single photon emission tomography (SPECT) of radiolabeled neurochemical tracers. Among the possible biochemical markers of specific neurons and synapses, including neurotransmitter synthetic enzymes, plasma membrane uptake transporters, and neurotransmitter receptors, a quantitative relationship between neuron or synapse integrity and the level of marker expression in macroscopic tissue samples is difficult to establish. The activities and expressed protein levels of these markers are well recognized to undergo compensatory regulation in response to therapeutic drugs and in response to disease conditions. Recent molecular and pharmacologic advances now permit the study of an additional class of neuronal markers, the distinct vesicular neurotransmitter transporters Advancer in Pharmacology, Volume 42 Copyright (C 1998 by Academic Press. All rights ot reproduction in any form reserved 1054-3589/98 $25.00
269
270
Kirk A. Frey et a/.
in monoaminergic and cholinergic neurons. Although neurotransmitter release from synaptic vesicles is a regulated process, it has been suggested that a major aspect of this regulation may reside in the partitioning of vesicles between active (cycling) and reserve (bound) pools in nerve terminals (1).We hypothesized that vesicular markers not distinguishing these states might serve as quantitative indicators of synaptic density, with minimal or no influence of regulatory changes. Measures of the vesicular neurotransmitter transporters may, thus, provide unique insights into conditions that alter synaptic regulation versus neuronal integrity. We synthesized both unlabeled and radioligand series targeting the neuronal vesicular monoamine transporter type-2 (VMAT2)or the vesicular acetylcholine transporter (VAChT). Ligand-binding specificities were established in vitro in direct binding and competition assays and after selective lesions of dopaminergic or cholinergic projection pathways. The possible regulation of VMAT2 and of VAChT by drugs that alter synaptic transmission were also studied. Single photon- or positron-emitting ligand analogues were then developed for in vivo SPECT and PET imaging of VMAT2 and VAChT. In vitro studies identified specificVMAT2 binding either with [3H]methoxytetrabenazine (2) or with stereochemically resolved ( +)-[3H]dihydrotetrabenazine (DTBZ). Binding of both ligands was saturable, reversible, and of high affinity ( K ~ 4s nM and 1.5 nM, respectively) to an apparently homogeneous population of binding sites on intact, slide-mounted brain sections. The brain regional pattern of binding was consistent with the known distributions of dopaminergic, noradrenergic, and serotonergic neurons and their projections. After 6-hydroxydopamine lesions, linear correlation of striatal VMAT2 binding and nigral tyrosine hydroxylase-immunoreactive neuron density was observed over a wide range of lesion severity (2). Furthermore, VMAT2 binding was not affected by 2-wk pretreatment of animals with a variety of drugs leading to regulatory changes in dopamine receptors or in the synaptic membrane dopamine transporter ( 3 ) .In vivo studies in rodents revealed cerebral biodistributions of the tracers paralleling the in vitro pattern of specific VMAT2 binding and additionally confirmed lack of interfering effects of nonvesicular ligand, dopaminergic drugs. Chromatographic analysis after the injection of DTBZ revealed only unchanged tracer in brain, permitting use of total tissue tracer levels in subsequent tracer kinetic analyses. Human imaging experiments employing PET and ["CIDTBZ revealed separable effects of tracer delivery (blood-to-brain transport) and VMAT2 binding on the cerebral time courses of activity after intravenous injection (4). We detected significant age-associated losses of striatal VMAT2 binding in normal subjects, corresponding to linear 0.8% per year losses of VMAT2 from the putamen. Patients with PD demonstrated substantial reductions in VMAT2 in the putamen and more minor losses in the caudate nucleus in the early stages. Patients with severe, advanced PD demonstrated near-complete losses of VMAT2 binding throughout the striatum. Patients with multiple-systems atrophy and some patients with sporadic olivopontocerebellar atrophy also demonstrate reduced striatal VMAT2 binding, supporting prior hypotheses of phenotypic overlap between these two conditions.
Imaging of Vesicular Transporters in the Brain
27 I
Initial in vitro analyses of the VAChT, based on [3H]vesamicol binding, revealed multiple sites of interaction for the ligand. Specific VAChT binding was identified for benzovesamicol-derivative ligands, including methylaminobenzovesamicol (MABV) and 5-iodobenzovesamicol (IBVM). Similarly to the VMAT2, VAChT expression was not regulated by drug treatments affecting ACh turnover and release. After fornix lesions, virtually complete loss of hippocampal VAChT binding was observed in in vitro VAChT binding assays. Intravenous injections of ['*'I]IBVM led to initial uptake in proportion to cerebral blood flow, but delayed retention in a pattern consistent with the in vitro distribution of VAChT, including heavy labeling of cholinergic nuclei and projections ( 5 ) .Fimbrial lesion reduced in vivo retention of ['2SI]IBVMto background levels, confirming the specificity of IBVM biodistribution. Human brain imaging of VAChT in vivo was developed on the basis of SPECT employing ['231]IBVM.After intravenous injections, tracer entered the brain readily and continued to rise over 12-18 hr in the striatum. The brain regional tracer distribution at 24 hr postinjection paralleled the VAChT distribution observed in in vitro assays. Tracer kinetic analyses confirmed close correlation of in vivo ['231]IBVMbinding to VAChT and the levels of delayed tracer retention, permitting simplified experimental determinations of immediate tracer uptake and 24-hr delayed retention in subsequent clinical studies of aging and dementing disorders. In contrast to striatal VMAT2, cerebral VAChT binding was not significantly affected by aging. Unexpectedly, however, VAChT binding showed relatively minor losses in the cerebral cortex of patients with AD ( 6 ) ,despite extensive prior reports of reduced cortical presynaptic cholinergic markers such as choline acetyltransferase (CAT)and acetylcholinesterase activities. There are apparent distinctions between AD patients with presenile versus senile onset of symptoms, the former demonstrating apparently greater VAChT reductions. In addition, PD patients with dementia reveal reduced cortical VAChT, in keeping with postmortem findings of concomitant AD changes in approximately half of demented PD patients. We have confirmed the SPECT VAChT findings in postmortem in vitro assays of AD (7).Samples of temporal neocortex reveal nonsignificant, approximately 15% losses of VAChT binding, yet have over 40% losses of CAT activity in parallel assays. Both VAChT and CAT are significantly reduced in the hippocampal formation of AD, but again, the VAT reduction is less than half of the magnitude of CAT change. These findings suggest the possibility of persistent cholinergic nerve terminals in AD cortex, but with reduced or absent CAT expression. Surviving cholinergic terminals may, thus, provide an attractive target substrate for the possible treatment of the cholinergic lesion in AD with neurotrophic factors that might restore CAT and other obligatory transmitter synthetic activities. In summary, vesicular neurotransmitter transporters appear to provide measures of presynaptic cholinergic and monoaminergic neuronal integrity, unaffected by use- or drug-induced regulatory changes. The roles of cholinergic and monoaminergic neurons in neurodegenerative diseases and their responses to potential disease-modifying therapies can now be specifically studied in vivo with PET and SPECT measures.
272
Kirk A. Frey et 01.
Acknowledgments The studies reported in this work were supported in part by U.S. Public Health Service grants AGO8671 and MH47611 from the National Institute of Aging and the National Institute of Mental Health, respectively.
References 1 . Greengard, P., Valtorta, F., Czernik, A. J., and Benfenati, F. (1993).Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 780. 2. Vander Borght, T. M., Sima, A. A. F., Kilbourn, M. R., Desmond, T. J., Kuhl, D. E., and Frey, K. A. (1995). [’HIMethoxytetrabenazine: A high specific activity ligand for estimating monoaminergic neuronal integrity. Neuroscience 68, 995-962. 3. Vander Borght, T. M., Kilbourn, M. R., Desmond, T. J., Kuhl, D. E., and Frey, K. A. The vesicular monoamine transporter is not regulated by dopaminergic drug treatments. Eur. J. Pharmacol. 294, 577-583. 4. Frey, K. A., Koeppe, R. A., Kilbourn, M. R., Vander Borght, T. M., Albin, R. L., Gilman, S., and Kuhl, D. E. (1996). Presynaptic monoaminergic vesicles in Parkinson’s disease and normal aging. Ann. N e w o l . 40, 873-884. 5 . Jung, Y-W., Frey, K. A., Mulholland, G. K., del Rosario, R., Sherman, P. S., Raffel, D. M., Van Dort, M. E., Kuhl, D. E., and Wieland, D. M. (1996). Vesamicol receptor mapping of brain cholinergic neurons with radioiodine labeled positional isomers of benzovesamicol. J. Med. G e m . 39, 3331-3342. 6. Kuhl, D. E., Minoshima, S., Fessler, J. A., Frey, K. A., Foster, N. L., Ficaro, E. P., Wieland, D. M., and Koeppe, R. A. (1996).In vivo mapping of cholinergic terminals in normal aging, Alzheimer’s disease and Parkinson’s disease. Ann. Neurol. 40, 399-410. 7. Murrnan, D. L., Desrnond, T. J., Higgins, D. S., Hermanowicz, N. S., Penney, J. B. Jr., and Frey, K. A. (1994).Autoradiographic quantification of the vesamicol receptor in Alzheimer’s disease. Neurology 44, (Suppl. 2), A389.